Particular blood cells, for example tumor cells or trophoblasts, are present in very small proportions and must be counted prior to conducting cytological analyses.
It is known that a formaldehyde-based binding buffer is to be applied to a blood sample in order to fix the desired cells, and the resulting liquid then passed through a porous filter. This filter is then used to examine the desired cells on it under a microscope in a laboratory. However, it is not possible to obtain live cells using this procedure.
Such being the case, the inventor has determined that obtaining live cells would make it possible to identify specific markers and, under good conditions, to apply molecular biology, cytogenetic and FISH (the acronym for “Fluorescence In Situ Hybridization”) techniques in diagnosing genetic abnormalities in tumor or trophoblastic cells.
The purpose of the present invention is to remedy these disadvantages and to meet this requirement by making it possible, under conditions compatible with standard laboratory testing, to collect live cells which can subsequently be cultivated in appropriate media in the presence of suitable growth factors.
The present invention also relates to the extraction of genetic material amplified if necessary from cells isolated on a filter and the detection of variations and gene expression levels for sensitivity and resistance to target treatments or for genetic abnormalities.
It applies in particular to the collection and potential uniform amplification of the DNA or RNA of particular cells present in a liquid, especially blood.
It is known, for example from the document PCT/FR 2006/000562, that a formaldehyde-based binding buffer is to be applied to a blood sample to fix the desired cells being sought, and the resulting liquid then passed through a porous filter. This filter is then analyzed under a microscope in a laboratory to detect the cells on it. The cells can then be sampled on the filter for analysis, for example by genetic analysis.
However, this procedure cannot be reproduced on a large scale at a reasonable cost, owing to the time, materials and precision of the work involved. Such being the case, reproduction on a large scale and at a reduced cost would enable molecular biology analyses to be conducted both on tumor cells and trophoblastic cells. Furthermore, fixing cells with formaldehyde does not allow good quality genetic material to be obtained: the DNA is partially degraded and forms bridges with ambient proteins and RNA extraction is virtually ruled out.